Carboxymethylcellulase isolated from bacillus sp. PKM-5430 (FERM BP-4087)

ABSTRACT

Carboxymethylcellulases 5430 and 5812 which show strong activity toward carboxymethylcellulose and lichenan, and moreover are broad in active pH range and active temperature range, stable in the presence of various surfactants and excellent in thermal stability and particularly, in alkali resistance. These enzymes can be advantageously used as formulation components for detergent compositions. The carboxymethylcellulases are isolated from Bacillus strains FERM BP-4087 and FERM BP-4088. Some of the characteristics of the enzymes include: a molecular weight of 26,000±1,000 as estimated by gel filtration on Bio-Gel A 0.5 m, the ability to hydrolyze carboxymethylcellulose, two optimum pH values of about 7.7 and 9.5, and a temperature optimum at about 55° C.

TECHNICAL FIELD

The present invention relates to new carboxymethylcellulases, and morespecifically to new enzymes, carboxymethylcellulases, which are broad inactive pH range and active temperature range and particularly, farexcellent in pH stability and thermal stability, and microorganismsproducing such enzymes.

BACKGROUND ART

Cellulases consist of a complex enzyme system which catalyzes anenzymatic reaction, in which celluloses and analogous polysaccharidesthereof are hydrolyzed to glucose, or cellobiose orcello-oligosaccharides, and are understood to be a general term forenzymes referred to as C₁ enzymes, C_(x) enzymes, β-glucosidases,exo-β-glucanases, endo-β-glucanases, cellobiases or the like accordingto their action mechanisms. Viewed from a historic light, the researchof the cellulases has been devotedly advanced for a long time with aview toward putting biomass resources to good use. Fungi, for example,the genera Trichoderma, Aspergillus, Acremonium and Humicola have beenrequired for sources of their supply. However, cellulases frommicroorganisms including fungi have specificity on action of theirconstitutive enzyme groups and diversity on their physicochemicalproperties and the like. Therefore, their actual conditions can be yethardly said to have been made clear.

Among the cellulases, those particularly high in action oncarboxymethylcellulose, i.e., endo-type hydrolytic action, are calledcarboxymethylcellulases. In recent years, an application to, forexample, an additive for laundry detergents has been developed as a newindustrial use for the cellulases including carboxymethyl-cellulases.Judging from the above-described cellulases from microorganisms,however, most of cellulases produced from microorganisms in naturalworld show optimum activities at acidic pHs and are deactivated atalkaline pHs. Namely, they are acidic cellulases (optimum active pH:4-6). Under the circumstances, there has been found only few alkalinecellulases and alkali-resistant cellulases, suitable for use in laundrydetergent which gives alkaline pHs.

The term "alkaline cellulase" used herein is intended to mean one whoseoptimum pH is in an alkaline range, and the term "alkali-resistantcellulase" means one whose optimum pH is in a neutral to acidic range,but which has a satisfactory activity in an alkaline region as comparedwith an activity at an optimum pH and is maintained stable.

Namely, there have heretofore been reported only, as processes for theproduction of alkaline cellulases and alkali-resistant cellulases, whichcan be used in laundry detergent compositions, a process in which analkaliphilic species of the genus Bacillus is incubated to collectCellulase A (Japanese Patent Application Laid-Open No. 28515/1975), aprocess in which an alkaliphilic species belonging to the genusCellulomonas is incubated to produce alkaline Cellulase 301-A (japanesePatent Application Laid-Open No. 224686/1983), a process in whichalkaliphilic Bacillus No. 1139 is incubated to produce acarboxymethylcellulase [F. Fukumori, T. Kudo and K. Horikoshi, J. Gen.Microbiol., 131, 3339(1985)], and a process in which a species of thegenus Streptomyces is used to produce an alkaline cellulase (JapanesePatent Application Laid-Open No. 19483/1986). None of these processeswere suitable for use in industrial production by fermentation.

On the other hand, it has recently been reported that Bacillus sp.KSM-635 (FRI Deposition No. 8872), which is a species of alkaliphilicmicroorganisms, effectively produces a carboxymethylcellulase suitablefor use in laundry detergent compositions and moreover, thecarboxymethylcellulase can be provided with higher yield by suitablyselecting incubation conditions, thereby permitting its commercial andindustrial production by fermentation.

However, the carboxymethylcellulase from the above-described Bacillussp. KSM-635 shows an optimum active temperature of about 40° C. and issuitable for low-temperature washing. On the other hand, there is ademand for an enzyme capable of using in laundry detergents at highertemperatures. In addition, it is desired that a carboxymethylcellulasehaving high stability at higher pH values should be found because thedetergency of detergent generally becomes enhanced at higher pH values.

Therefore, it is an object of this invention to provide acarboxymethylcellulase capable of acting even at high temperatures,broad in active pH range and in particular, good in stability at hightemperatures and high pHs.

The present inventors have continued an extensive search with a viewtoward finding microorganisms capable of producing acarboxymethylcellulase from the natural world. As a result, it has beenfound that microorganisms collected from the soil in The Philippines andbelonging to the genus Bacillus produce new Carboxymethylcellulase 5430or 5812, which is useful as an additive for laundry detergentcompositions, and Carboxymethylcellulases 5430 and 5812 are broader inactive pH range than the conventional carboxymethylcellulases, excellentin thermal stability and in particular, good in stability at high pHvalues, leading to completion of the present invention.

DISCLOSURE OF THE INVENTION

The present invention is directed to Carboxymethylcellulases 5430 and5812 broad in active pH range and active temperature range, good inthermal stability and excellent in resistance to alkali.

The present invention is also concerned with microorganisms belonging tothe genus Bacillus and producing said Carboxymethylcellulase 5430 or5812.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1, 2, 3 and 4 illustrate a pH-activity curve ofCarboxymethylcellulase 5430, a pH stability curve ofCarboxymethylcellulase 5430, a temperature-activity curve ofCarboxymethylcellulase 5430 and a temperature stability ofCarboxymethylcellulase 5430, respectively.

FIGS. 5, 6, 7 and 8 illustrate a pH-activity curve ofCarboxymethylcellulase 5812, a pH stability curve ofCarboxymethylcellulase 5812, a temperature-activity curve ofCarboxymethylcellulase 5812 and a temperature stability ofCarboxymethylcellulase 5812, respectively.

BEST MODE FOR CARRYING OUT THE INVENTION

Microorganisms producing Carboxymethylcellulase 5430 or 5812 accordingto this invention show the following bacteriological properties.Incidentally, the following A through C are media used for isolating andincubating the enzyme-producing bacteria. Besides, Na₂ CO₃ wassterilized separately from other components to add it.

Medium A (PCMC medium):

polypeptone (product of Nippon Seiyaku K.K), 20 g;carboxymethylcellulose (CMC, product of Sanyo Kokusaku Pulp Co., Ltd.),10 g; yeast extract (product of Difco Laboratories), 1 g; KH₂ PO₄(product of Wako Pure Chemical Industries, Ltd.), 1 g; NaCl (product ofMerck), 5 g; Na₂ CO₃ (anhydrous; product of Iwai Kagaku Co., Ltd.) 5 g;MgSO₄ 7H₂ O (product of Wako Pure Chemical Industries, Ltd.), 0.2 g;Trypan blue dye (product of Merck), 0.075 g; agar (product of DifcoLaboratories), 15 g (deionized water was added to prepare a 1.0-1medium).

Medium B:

Prepared in the same manner as in Medium A except for omission of Trypanblue dye.

Medium C (CMC-peptone medium):

CMC, 10 g; polypeptone, 20 g; yeast extract, 0.5 g; KH₂ PO₄, 1 g; NaCl,5 g; Na₂ CO₃, 5 g (deionized water was added to prepare a 1.0-medium).

Bacteriological Properties of Carboxymethylcellulase 5430-ProducingBacteria

1. Result of microscopic observation:

The microorganisms are rods of 0.7-0.8 μm×1.2-2.9 μm in size, which formellipsoidal spores (0.7-1.0 μm×0.7-1.5 μm) on the center or subterminalsthereof.

2. Motility: Positive, with peritrichous flagella.

3. Gram-staining: Positive.

4. Growth conditions in various media:

(1) On nutrient agar plate medium:

The shape of colony is circular. Its surface is rough, and the edge isundulate. The color of the colony is white.

(2) In nutrient broth medium:

Grown.

(3) NaCl tolerance in nutrient broth:

The bacteria grow in 5% and 7% NaCl, but do not grow in 10% NaCl.

(4) On gelatin medium:

Gelatin is liquefied.

(5) In litmus milk medium:

The upper layer of the culture medium is slightly decolored.

5. Physiological properties:

(1) Reduction of nitrate to nitrite: Negative.

(2) Denitrification: Negative.

(3) MR test: Negative.

(4) V-P test: Positive.

(5) Formation of indol: Negative.

(6) Formation of hydrogen sulfide: Positive.

(7) Hydrolysis of starch: Negative.

(8) Hydrolysis of casein: Positive.

(9) Utilization of citrate:

Simons citrate agar plate medium: Positive.

Christensen citrate agar plate medium: Positive.

(10) Utilization of inorganic nitrogen: Negative.

(11) Formation of pigment:

King A medium: Negative.

King B medium: Negative.

Potato Dextrose Agar Eiken: Negative.

Mannitol-Salt Agar medium: Negative.

1(12) Urease: Negative.

(13) Oxidase: Positive.

(14) Catalase: Positive.

(15) Range of growth:

Temperature for growth: 15°-50° C.

Optimum temperature for growth: 25°-<50° C.

pH for growth: 4.7-10

Optimum pH for growth: 6.0-9.5

(16) Requirement for Oxygen:

Grown both aerobically and anaerobically.

(17) O-F test: Fermented.

(18) Utilization of propionate: Positive.

(19) Degradation of tyrosine: Negative.

(20) Deamination of phenylalanine: Negative.

(21) Acid formation from sugars:

Positive: D-ribose, L-arabinose, D-xylose, D-glucose, D-mannose,sucrose, trehalose, mannitol, glycerol, D-galactose, sorbitol.

Negative: D-fructose, maltose, inositol, lactose, dextrin, raffinose,starch.

(22) G+C content of DNA: 42.9 mol %.

Bacteriological Properties of Carboxymethylcellulase 5812-ProducingBacteria

1. Result of microscopic observation:

The microorganisms are rods of 0.6-0.8 μm×1.5-3.3 μm in size, which formellipsoidal spores (0.7-1.0 μm×1.0-1.6 μm) on the center or subterminalsthereof.

2. Motility: Positive, with peritrichous flagella.

3. Gram-staining: Positive.

4. Growth conditions in various media:

(1) On nutrient agar plate medium:

The shape of colony is circular. Its surface is rough, and the edge isundulate. The color of the colony is white or extremely pale yellow.

(2) In nutrient broth medium:

Grown.

(3) NaCl tolerance in nutrient broth:

The bacteria grow in 5% NaCl, but do not grow in 7% and 10% NaCl.

(4) On gelatin medium:

Gelatin is liquefied.

(5) In Litmus milk medium:

The upper layer of the culture medium is slightly decolored.

5. Physiological properties:

(1) Reduction of nitrate to nitrite: Negative.

(2) Denitrification: Negative.

(3) MR test: Negative.

(4) V-P test: Positive.

(5) Formation of indol: Negative.

(6) Formation of hydrogen sulfide: Positive.

(7) Hydrolysis of starch: Negative.

(8) Hydrolysis of casein: Positive.

(9) Utilization of citrate:

Simons citrate agar plate medium: Positive.

Christensen citrate agar plate medium: Positive.

(10) Utilization of inorganic nitrogen: Negative.

(11) Formation of pigment:

King A medium: Negative.

King B medium: Negative.

Potato Dextrose Agar Eiken: Negative.

Mannitol-Salt Agar medium: Negative.

(12) Urease: Negative.

(13) Oxidase: Positive.

(14) Catalase: Positive.

(15) Range of growth:

Temperature for growth: 15°-45° C.

Optimum temperature for growth: 25°-<45° C.

pH for growth: 4.7-10

Optimum pH for growth: 6.0-9.5

(16) Requirement for Oxygen:

Grown both aerobically and anaerobically.

(17) O-F test: Fermented.

(18) Utilization of propionate: Positive.

(19) Degradation of tyrosine: Negative.

(20) Deamination of phenylalanine: Negative.

(21) Acid formation from sugars:

Positive: D-ribose, L-arabinose, D-xylose, D-glucose, mannitol.

Negative: D-mannose, D-fructose, maltose, sucrose, trehalose, inositol,glycerol, D-galactose, lactose, sorbitol, dextrin, raffinose, starch.

(22) G+C content of DNA: 42.6 mol %.

The above-described bacteriological properties were referred to Bergey'sManual of Systematic Bacteriology, Vol. 2. As a result, these strainswere judged to be species of the genus Bacillus, which were rods withspore.

Since the bacteriological properties of these strains differed fromthose of any known Bacillus species, these strains were judged to be newstrains, named "Bacillus sp. PKM-5430" and "Bacillus sp. PKM-5812", anddeposited with the Fermentation Research Institute, Agency of IndustrialScience and Technology as International Deposition Nos. BP-4087 andBP-4088, respectively.

In order to collect Carboxymethylcellulase 5430 or 5812 according tothis invention, it is only necessary to incubate the above-describedBacillus sp. PKM-5430 or PKM-5812 or a variant thereof in a medium, andisolate and purify the resulting culture by the conventionally-knownpurification method for enzyme.

Upon the fermentative production of Carboxymethylcellulase 5430, it isonly necessary to sterilize a suitable medium by heating or the like andthen inoculate Bacillus sp. PKM-5430 (International Deposition No.BP-4087) into the medium to subject it to shaking culture or aerobicspinner culture for 1-4 days at 15°-50° C., preferably 25°-45° C. Theadjustment of pH to neutrality to 10 gives good results. Althoughfoaming may occur in some cases, this problem is solved by suitablyadding an appropriate antifoam to the medium.

Upon the fermentative production of Carboxymethylcellulase 5812 on theother hand, it is only necessary to sterilize a suitable medium byheating or the like and then inoculate Bacillus sp. PKM-5812(International Deposition No. BP-4088) into the medium to subject it toshaking culture or aerobic spinner culture for 1-4 days at 15°-45° C.,preferably 25°-43° C. The adjustment of pH to weak acidity (pH: about 5)to 10 gives good results. Although foaming may occur in some cases, thisproblem is solved by suitably adding an appropriate antifoam to themedium.

Carboxymethylcellulase 5430 or 5812 may be produced by incorporatingassimilable nitrogen source and carbon source in suitable combinationinto a culture medium. No particular limitation is imposed on bothnutrient sources. As exemplary nitrogen sources, may be mentionedorganic nitrogen sources such as corn gluten meal, soybean powder, cornsteep liquor, casamino acids, yeast extract, Pharmamedia, sardine meal,meat extract, peptone, Hipro, Ajipower, coffee cake, cottonseed cake,Cultivator, Amiflex, Ajipron, Zest and Ajix. As illustrative carbonsources, may be mentioned chaff, wheat-gluten bread, vegetable fibrousmaterials such as filter paper and sawdust, blackstrap molasses, invertsugar, CMC, Avicel, cellulose cotton, xylan, pectin, and in addition,assimilable carbon sources, for example, ribose, arabinose, xylose,glucose, mannose, fructose, maltose, sucrose, mannitol, inositol andglycerol, and assimilable organic acids, for example, acetic acid andcitric acid. Namely, any medium obtained by suitably combining thesenitrogen sources and carbon sources with each other may be used. Theabove-mentioned nutrient sources are not particularly limited. Inaddition, media containing phosphoric acid, inorganic salts such asMg²⁺, Ca²⁺, Mn²⁺, Zn²⁺, Ca²⁺, Na⁺ and K⁺ and optionally inorganic andorganic minor nutrient sources may be suitably chosen for use.

In order to obtain Carboxymethylcellulase 5430 or 5812 from thethus-obtained culture, the general collection and purification means maybe followed. Namely, the culture is centrifuged or filtered to separatethe strain from the culture. The supernatant of the culture are treatedby the conventional separation means, for example, the salting out,isoelectric precipitation or solvent precipitation (methanol, ethanol,isopropanol, etc.), to precipitate proteins, or concentrated byultrafiltration, thereby obtaining a crude enzyme solution ofCarboxymethylcellulase 5430 or 5812. After precipitating the enzyme in,for example, ammonium sulfate (30-100% saturated fraction) for thesalting out, or in, for example, 75% ethanol for the solventprecipitation, the precipitate may be filtered or centrifuged anddesalted to form lyophilized powder. As a method for the desalting, isused a general method such as dialysis or gel filtration on Sephadex orBio-Gel.

Although the crude enzyme solution may be used as it is, as necessary,it may also be purified by the conventionally-known method, for example,the suitable combination of chromatography on hydroxyapatite,ion-exchange chromatography on DEAE-Sephadex or DEAE-Bio-Gel and gelfiltration chromatography on Sephadex or Bio-Gel to use it as a purifiedenzyme.

The thus-obtained Carboxymethylcellulases 5430 and 5812 (crude enzymes)have such properties as described below. Incidentally, the determinationof enzymatic activities was conducted in accordance with the followingmethods.

(a) Carboxymethylcellulase activity:

Activities were determined by the DNS method (J. R. Summer and G. F.Somers, Laboratory Experiments in Biological Chemistry, Academic Press,New York, U.S.A., 1944). Namely, 0.1 ml of a suitably diluted enzymesolution was added to 0.9 ml of a 1.1% (w/v) solution of a CMC (A01MC)substrate in 0.1M glycine-NaOH buffer (pH: 9.0). After a 20-minutereaction at 40° C., 1 ml of 0.05% (w/v) 3,5-dinitrosalicylic acidsolution (product of Wako Pure Chemical Industries, Ltd.) was added, andthe resulting mixture was immediately placed in a boiling water bath for5 minutes, followed by cooling in an iced water bath. Then, 4 ml ofdeionized water was added further, afterwhich, colorimetry was conductedby measuring an absorbance at 535 nm. With respect to enzymaticactivity, the amount of enzyme, which liberates reducing sugarscorresponding to 1 μmole of glucose for 1 minute, was defined as 1 unit.

(b) Hydrolytic activities toward cellulosic substrates:

Sodium hydroxide- and phosphoric acid-swollen celluloses were preparedaccording to the method of Tomita et al. [Y. Tomita, H. Suzuki and K.Nisizawa, J. Biochem., 78, 499(1975)]. The reducing sugar liberated wasdetermined in a similar manner to that of the method (a) except thatsubstrate solutions containing cellulose substrates were used, and thereaction was conducted at 30° C.

(c) Hydrolytic activities toward polysaccharides:

The reducing sugar liberated was determined in a similar manner to thatof the method (a) except that substrate solutions containingpolysaccharides were used, and the reaction was conducted at 30° C.

(d) Hydrolytic activities toward p-nitrophenyl-β-D-glucopyranoside(PNPG; product of Sigma Chemical) and p-nitrophenyl-β-D-cellobioside(PNPC; product of Sigma Chemical):

A suitably diluted enzyme solution was added to a solution of 8 mM of aPNPG or PNPC substrate in 0.1M phosphate buffer of pH 7.0 to react themfor 20 minutes at 30° C. The p-nitrophenol liberated was determined at400 nm and the amount formed was calculated.

Enzymological Properties of Carboxymethylcellulase 5430

(1) Action:

This enzyme has an enzymatic activity toward CMC.

(2) Substrate specificity:

As shown in the following Table 1, this enzyme shows strong activitytoward CMC and lichenan. The enzyme is also active toward cellobiose,PNPG, PNPC, cellulose powder and phosphoric acid-swollen cellulose.

                  TABLE 1                                                         ______________________________________                                                        Concentration                                                                              Relative                                         Substrate       of substrate activity (%)                                     ______________________________________                                        CMC*.sup.1      1.0%         100.0                                            Avicel          1.0%         0.0                                              Cellulose Powder                                                                              1.0%         0.1                                              (Sigmacell 20)                                                                Cellulose Powder                                                                              1.0%         1.1                                              (Sigmacell 100)                                                               Phosphoric acid-swollen                                                                       1.0%         7.3                                              cellulose                                                                     Sodium hydroxide-swollen                                                                      1.0%         0.0                                              cellulose                                                                     Curdlan*.sup.2  1.0%         0.0                                              Laminarin*.sup.3                                                                              1.0%         1.1                                              Lichenan*.sup.4 1.0%         78.7                                             Cellobiose      0.1%         0.4                                              PNPC            8 mM         1.0                                              PNPG            8 mM         0.8                                              ______________________________________                                         *.sup.1 : The activity toward CMC at 30° C. was taken as 100% to       determine relative activities toward other substrates.                        *.sup.2 : Derived from Alcaligenes faecalis var. myxogenes; product of        Wako Pure Chemical Industries, Ltd.                                           *.sup.3 : Derived from Laminaria digitata; product of Sigma Chemical.         *.sup.4 : Derived from Cetraria islandica; product of Sigma Chemical.    

(3) Active pH range and optimum pH:

This enzyme is active in a pH range of 4.2-12.5 and has two optimum pHvalues of about 7.7 and about 9.5 (FIG. 1). Even at pH 10, about 90% ofthe maximum activity is shown, while at pH 11.5, the activity is stillhigher than about 50%. Even at pH 12.5, about 20% of the activity ismaintained.

(4) pH Stability:

The stability of the enzyme to pH was investigated by overnighttreatment at 5° C. in Macllvaine buffer (pH: 3-7), 0.1Mtris-hydrochloride buffer (pH: 7-9), 0.1M glycine-sodium hydroxidebuffer (pH: 8-11) and 0.1M potassium chloride-sodium hydroxide buffer(pH: 9-14) and then measurement of residual activities in 0.1M sodiumphosphate buffer of pH 6.0 and 0.1M glycine-sodium hydroxide buffer ofpH 9.0. As a result, the enzyme is extremely stable over a pH range of4.6-12.8 on both pHs (FIG. 2).

(5) Active temperature range and optimum temperature:

The carboxymethylcellulase activity of this enzyme was measured in 0.1Mglycine-sodium hydroxide buffer of pH 9.0. The enzyme is active over awide temperature range of 10°-78° C., and its optimum temperature isobserved at about 55° C. (FIG. 3).

(6) Thermal stability:

After pretreating this enzyme for 10 minutes at various temperatures,its residual activity was determined in 0.1M glycine-sodium hydroxidebuffer of pH 9.0 to investigate the thermal stability thereof. As aresult, it was found that the enzyme was scarcely deactivated afterheated to 60° C., and still retained some activity even after heated to80° C. (FIG. 4). The addition of Ca²⁺ brought about no effect under theabove conditions.

(7) Effects of metal ions:

Each metal ion preparation shown in Table 2 and CMC were mixed with theenzyme solution and the carboxymethylcellulase activity of the enzymewas measured for 20 minutes at 40° C. in 0.1M glycine-sodium hydroxidebuffer of pH 9.0. Among the various metal ions tested, Hg²⁺, at 1 mM,inhibited the activity of the enzyme by about 75%. On the contrary,Co⁺², at 1 mM stimulated the activity of the enzyme by about 30% andMn⁺², at 1 mM, also stimulated the activity by about 50%. The othermetal ions shown in Table 2 showed moderate levels of either inhibitionor stimulation and metal ions other than the above showed no effect atall.

                  TABLE 2                                                         ______________________________________                                                                  Relative                                            Metal ions  Concentration (mM)                                                                          activity (%)                                        ______________________________________                                        None        --            100                                                 Ca.sup.2+   1             108                                                 Mg.sup.2+   1             108                                                 Mn.sup.2+   1             130                                                 Co.sup.2+   1             149                                                 Pb.sup.2+   1              81                                                 Zn.sup.2+   1              92                                                 Hg.sup.2+   1              25                                                 Cu.sup.2+   1              97                                                 Fe.sup.2+   1             105                                                 Fe.sup.3+   1             110                                                 Al.sup.3+   1             101                                                 Na.sup.+    50            121                                                 K.sup.+     50            116                                                 ______________________________________                                    

(8) Effects of surfactants:

Surfactants shown in Table 3 were separately mixed with the enzymesolution to measure the carboxymethylcellulase activity of the enzymefor 20 minutes at 40° C. in 0.1M glycine-sodium hydroxide buffer of pH9.0. As apparent from Table 3, the activity of this enzyme was eitherscarcely inhibited or stimulated by sodium dodecyl sulfate (SDS), sodiumlinear alkylbenzenesulfonate (LAS), sodium alkyl sulfate (AS), sodiumpolyoxyethylene alkyl sulfate (ES), sodium α-olefin sulfonate (AOS),sodium α-sulfonated fatty acid ester (α-SFE), fatty acid salt (soap) orpolyoxyethylene alkyl ether. In particular, SDS and the fatty acid saltstimulated the activity of the enzyme by 26% and 45%, respectively.

                  TABLE 3                                                         ______________________________________                                                                  Relative                                            Surfactants Concentration (%)                                                                           activity (%)                                        ______________________________________                                        None        --            100                                                 SDS         0.05          126                                                 LAS         0.05          105                                                 AS          0.05          123                                                 ES          0.05          102                                                 AOS         0.05          107                                                 α-SFE 0.05          116                                                 Fatty acid  0.05          145                                                 salt (soap)                                                                   Softanol 70H                                                                              0.05          107                                                 ______________________________________                                    

(9) Effects of inhibitors:

Inhibitors shown in Table 4 were separately mixed with the enzymesolution to measure the carboxymethylcellulase activity of the enzymefor 20 minutes at 40° C. in 0.1M glycine-sodium hydroxide buffer of pH9.0. N-Bromosuccinimide drastically inhibited the activity of thisenzyme by 97%, while phenylmethanesulfonyl fluoride moderately inhibitedthe enzyme by 24%. However, the activity of the enzyme was not inhibitedby the other inhibitors.

                  TABLE 4                                                         ______________________________________                                                      Concentration                                                                             Relative                                            Inhibitors    (mM)        activity (%)                                        ______________________________________                                        None          --          100                                                 N-Bromo-*     0.1          4                                                  succinimide                                                                   Sodium azide  1.0         118                                                 Hydroxylamine 1.0         133                                                 hydrochloride                                                                 Monoiodo acetic                                                                             1.0         117                                                 acid                                                                          4-Chloromercuri-                                                              benzoic acid  1.0         118                                                 Phenylmethane-                                                                              1.0          76                                                 sulfonyl fluoride                                                             ______________________________________                                         *: The enzyme was pretreated at pH 5.5 (50 mM acetate buffer).           

(10) Effects of chelating agents, thiol reagents, additives andproteinases:

Reagents shown in Table 5 were separately mixed with the enzyme solutionto measure the carboxymethylcellulase activity of the enzyme for 20minutes at 40° C. in 0.1M glycine-sodium hydroxide buffer of pH 9.0. Theactivity of this enzyme was somewhat inhibited by EDTA or EGTA. On theother hand, 2-mercaptoethanol and dithiothreitol, each added at 1 mM,stimulated the activity of the enzyme by 39% and 50%, respectively. Itwas not observed that the proteinases such as alkalase, maxatase,savinase and Protease API-21 inactivated the enzyme of this inventionunder this reaction conditions.

                  TABLE 5                                                         ______________________________________                                                                   Relative                                           Reagents       Concentration                                                                             activity (%)                                       ______________________________________                                        None           --          100                                                Zeolite        0.05%        99                                                EDTA           1 mM         71                                                EGTA           1 mM         66                                                o-Phenanthroline                                                                             1 mM         98                                                2-Mercaptoethanol                                                                            1 mM        139                                                Dithiothreitol 1 mM        150                                                Alkalase       0.01%       111                                                Maxatase       0.01%       105                                                Savinase       0.01%       107                                                Protease API-21                                                                              0.01%       123                                                ______________________________________                                    

(11) Molecular weight:

The molecular weight of this enzyme was estimated by gel filtration onBio-Gel A 0.5 m in accordance with the method of Shikata and Nisizawa[S. Shikata and K. Nisizawa, J. Biochem., 78, 499(1975)] and found to beabout 26,000±1,000.

Enzymological Properties of Carboxymethylcellulase 5812

(1) Action:

This enzyme has an enzymatic activity toward CMC.

(2) Substrate specificity:

As shown in the following Table 6, this enzyme shows strong activitytoward CMC and lichenan. The enzyme is also active toward cellobiose,PNPG, PNPC, cellulose powder and phosphoric acid-swollen cellulose.

                  TABLE 6                                                         ______________________________________                                                        Concentration                                                                              Relative                                         Substrate       of substrate activity (%)                                     ______________________________________                                        CMC*.sup.1      1.0%         100.0                                            Avicel          1.0%         0.0                                              Cellulose Powder                                                                              1.0%         0.1                                              (Sigmacell 20)                                                                Cellulose Powder                                                                              1.0%         1.0                                              (Sigmacell 100)                                                               Phosphoric acid-swollen                                                                       1.0%         7.5                                              cellulose                                                                     Sodium hydroxide-swollen                                                                      1.0%         0.0                                              cellulose                                                                     Curdlan*.sup.2  1.0%         0.0                                              Laminarin*.sup.3                                                                              1.0%         1.0                                              Lichenan*.sup.4 1.0%         71.2                                             Cellobiose      0.1%         0.4                                              PNPC            8 mM         0.6                                              PNPG            8 mM         0.2                                              ______________________________________                                         *.sup.1 : The activity toward CMC at 30° C. was taken as 100% to       determine relative activities toward other substrates.                        *.sup.2 : Derived from Alcaligenes faecalis var. myxogenes; product of        Wako Pure Chemical Industries, Ltd.                                           *.sup.3 : Derived from Laminaria digitata; product of Sigma Chemical.         *.sup.4 : Derived from Cetraria islandica; product of Sigma Chemical.    

(3) Active pH range and optimum pH:

This enzyme is active in a pH range of 4.2-12.5 and has two optimum pHvalues of about 7.7 and about 9.5 (FIG. 5). Even at pH 10, about 90% ofthe maximum activity is shown, while at pH 11.5, the activity is stillhigher than about 30%. Even at pH 12.5, about 10% of the activity ismaintained.

(4) pH Stability:

The stability of the enzyme to pH was investigated by overnighttreatment at 5° C. in Macllvaine buffer (pH: 3-7), 0.1Mtris-hydrochloride buffer (pH: 7-9), 0.1M glycine-sodium hydroxidebuffer (pH: 8-11) and 0.1M potassium chloride-sodium hydroxide buffer(pH: 9-14) and then measurement of residual activities in 0.1M sodiumphosphate buffer of pH 6.0 and 0.1M glycine-sodium hydroxide buffer ofpH 9.0. As a result, the enzyme is extremely stable over a pH range of4.8-12 on both pHs (FIG. 6).

(5) Active temperature range and optimum temperature:

The carboxymethylcellulase activity of this enzyme was measured in 0.1Mglycine-sodium hydroxide buffer of pH 9.0. The enzyme is active over awide temperature range of 10°-70° C., and its optimum temperature isobserved at about 55° C. (FIG. 7).

(6) Thermal stability:

After pretreating this enzyme for 10 minutes at various temperatures,its residual activity was determined in 0.1M glycine-sodium hydroxidebuffer of pH 9.0 to investigate the thermal stability thereof. As aresult, it was found that the enzyme was scarcely deactivated afterheated to 60° C., and still retained some activity even after heated to70° C. (FIG. 8). The addition of Ca²⁺ brought about no effect under theabove conditions.

(7) Effects of metal ions:

Each metal ion preparation shown in Table 7 and CMC were mixed with theenzyme solution and the carboxymethylcellulase activity of the enzymewas measured for 20 minutes at 40° C. in 0.1M glycine-sodium hydroxidebuffer of pH 9.0. Among the various metal ions tested, Hg²⁺, at 1 mM,inhibited the activity of the enzyme by about 75%. On the contrary,Co⁺², at 1 mM stimulated the activity of the enzyme by about 50%, andMn⁺², at 1 mM, also stimulated the activity by 53%. The other metal ionsshown in Table 7 showed moderate levels of either inhibition orstimulation and metal ions other than the above showed no effect at all.

                  TABLE 7                                                         ______________________________________                                                                  Relative                                            Metal ions  Concentration (mM)                                                                          activity (%)                                        ______________________________________                                        None        --            100                                                 Ca.sup.2+   1             108                                                 Mg.sup.2+   1             109                                                 Mn.sup.2+   1             153                                                 Co.sup.2+   1             150                                                 Pb.sup.2+   1              93                                                 Zn.sup.2+   1              96                                                 Hg.sup.2+   1              27                                                 Cu.sup.2+   1              98                                                 Fe.sup.2+   1             101                                                 Fe.sup.3+   1             104                                                 Al.sup.3+   1              97                                                 Na.sup.+    50            120                                                 K.sup.+     50            126                                                 ______________________________________                                    

(8) Effects of surfactants:

Surfactants shown in Table 8 were separately mixed with the enzymesolution to measure the carboxymethylcellulase activity of the enzymefor 20 minutes at 40° C. in 0.1M glycine-sodium hydroxide buffer of pH9.0. As apparent from Table 8, the activity of this enzyme was eitherscarcely inhibited or stimulated by sodium dodecyl sulfate (SDS), sodiumlinear alkylbenzenesulfonate (LAS), sodium alkyl sulfate (AS), sodiumpolyoxyethylene alkyl sulfate (ES), sodium α-olefin sulfonate (AOS),sodium α-sulfonated fatty acid ester (α-SFE), fatty acid salt (soap) orpolyoxyethylene alkyl ether. In particular, SDS and the fatty acid saltstimulated the activity of the enzyme by 21% and 23%, respectively.

                  TABLE 8                                                         ______________________________________                                                                  Relative                                            Surfactants Concentration (%)                                                                           activity (%)                                        ______________________________________                                        None        --            100                                                 SDS         0.05          121                                                 LAS         0.05           96                                                 AS          0.05          112                                                 ES          0.05          112                                                 AOS         0.05          105                                                 α-SFE 0.05           97                                                 Fatty acid  0.05          123                                                 salt (soap)                                                                   Softanol 70H                                                                              0.05          116                                                 ______________________________________                                    

(9) Effects of inhibitors:

Inhibitors shown in Table 9 were separately mixed with the enzymesolution to measure the carboxymethylcellulase activity of the enzymefor20 minutes at 40° C. in 0.1M glycine-sodium hydroxide buffer of pH9.0. N-Bromosuccinimide drastically inhibited the activity of thisenzyme by 97%. However, the activity of the enzyme was not inhibited bythe other inhibitors.

                  TABLE 9                                                         ______________________________________                                                      Concentration                                                                             Relative                                            Inhibitors    (mM)        activity (%)                                        ______________________________________                                        None          --          100                                                 N-Bromo-*     0.1          3                                                  succinimide                                                                   Sodium azide  1.0         117                                                 Hydroxylamine 1.0         123                                                 hydrochloride                                                                 Monoiodo acetic                                                                             1.0         104                                                 acid                                                                          4-Chloromercuri-                                                                            1.0         105                                                 benzoic acid                                                                  Phenylmethane-                                                                              1.0         107                                                 sulfonyl fluoride                                                             ______________________________________                                         *: The enzyme was pretreated at pH 5.5 (50 mM acetate buffer).           

(10) Effects of chelating agents, thiol reagents, additives andproteinases:

Reagents shown in Table 10 were separately mixed with the enzymesolution to measure the carboxymethylcellulase activity of the enzymefor 20 minutes at 40° C. in 0.1M glycine-sodium hydroxide buffer of pH9.0. The activity of this enzyme was somewhat inhibited by EDTA or EGTA.On the other hand, 2-mercaptoethanol and dithiothreitol, each added at 1mM, stimulated the activity of the enzyme by 52% and 84%, respectively.It was not observed that the proteinases such as alkalase, maxatase,savinase and Protease API-21 inactivated the enzyme of this inventionunder this reaction conditions.

                  TABLE 10                                                        ______________________________________                                                                  Relative                                            Reagents       Concentration                                                                            activity (%)                                        ______________________________________                                        None           --         100                                                 Zeolite        0.05%      100                                                 EDTA           1 mM        79                                                 EGTA           1 mM        73                                                 o-Phenanthroline                                                                             1 mM       100                                                 2-Mercaptoethanol                                                                            1 mM       152                                                 Dithiothreitol 1 mM       184                                                 Alcalase       0.01%      107                                                 Maxatase       0.01%      109                                                 Savinase       0.01%      106                                                 Protease API-21                                                                              0.01%      110                                                 ______________________________________                                    

(11) Molecular weight:

The molecular weight of this enzyme was estimated by gel filtration onBio-Gel A 0.5 m in accordance with the method of Shikata and Nisizawa[S. Shikata and K. Nisizawa, J. Biochem., 78, 499(1975)] and found to beabout 26,000±1,000.

As described above, Carboxymethylcellulase 5430 features that it isactive over a very broad pH range of 4.2-12.5 and extremely stable atany pH up to 12.8, and moreover that it is active over a broadtemperature range of 10°-78° C., good in thermal stability and has thepotential for application to detergents formulated for high temperaturewashing and/or alkaline detergents. Also, Carboxymethylcellulase 5812features that it is active over a very broad pH range of 4.2-12.5 andextremely stable at any pH up to 12, and moreover that it is active overa broad temperature range of 10°-70° C., good in thermal stability andhas the potential for application to detergents formulated for hightemperature washing and/or alkaline detergents.

Further, the activities of these Carboxymethylcellulases 5430 and 5812are scarcely inhibited even by formulation ingredients for detergents,such as surfactants, metal ions and proteinases.

EXAMPLES

The present invention will hereinafter be described more specifically bythe following examples.

Example 1

About 0.5 g of a soil sample, which had been collected in Davao City,Davao der Sur, The Philippines, was suspended in 9 ml of a sterilephysiological saline solution and heated for 10 minutes at 80° C. Thethus-heat-treated soil sample was suitably diluted, spread on an agarplate [PCMC medium (Medium A)] and then incubated for 3 days at 30° C.After the incubation, colonies with clear zones were spread on the sameagar plate to incubate them for 3 days at 30° C., thereby formingcolonies again.

Bacillus sp. PKM-5430 (International Deposition No. BP-4087) accordingto this invention was obtained in accordance with the above-describedprocedure.

Example 2

A portion of Bacillus sp. PKM-5430 obtained in Example 1 was subjectedto slant culture using Medium B (prepared in the same manner as inMedium A except for omission of Trypan blue dye). Colonies thus obtainedwere then inoculated into 5 ml of a CMC-peptone medium (Medium C)contained in a 50-ml test tube and incubated for 2 days at 30° C. whileshaking at 300 rpm. A 1-ml portion of the above culture was furtherinoculated into the 50-ml portion of the Medium C contained in a 500-mlSakaguchi flask and incubated for 3 days at 30° C. while shaking at 120rpm.

Cells were removed from the culture medium thus obtained bycentrifugation to obtain a supernatant containing Carboxymethylcellulase5430.

Example 3

The supernatant obtained in Example 2 was treated with ammonium sulfate(pH was kept neutral with dilute aqueous ammonia), and proteins whichprecipitated at 90% saturation were collected by centrifugation(11,000×g). The precipitate was dissolved in 20 mM phosphate buffer ofpH 7.0, and dialyzed overnight at 5° C. against the same buffer.

The concentration of the proteins was determined by means of a Bio-Radprotein assay kit (product of Bio-Rad Laboratories) making use of bovineplasma gamma globulin as a standard in accordance with the method ofBradford [M. Bradford, Anal. Biochem., 72, 248(1976)], whereby a crudeenzyme solution having a specific activity of 3.47 unit (U)/mg ofprotein was found to be obtained.

Example 4

With respect to the crude enzyme solution obtained in Example 3,polyacrylamide gel electrophoresis (PAGE) was carried out on 9% (w/v)polyacrylamide slab gel (70 mm×80 mm, 1.0 mm thickness) making use of150 mM tris-glycine buffer (pH: 8.8) as a running buffer in accordancewith the method of Davis [B. J. Davis, Ann. N. Y. Acad. Sci., 121,404(1964)]. The electrophoresis was performed at 10 mA per gel for about2 hours at room temperature. The proteins were then visualized by silverstaining (kit of silver stain, product of Kanto Chemical Co., Inc.) inaccordance with the method of Oakley et al. [B. R. Oakley, D. R. Kirschand N. R. Morris, Anal. Biochem., 105, 361(1980)].

Protein bands associated with carboxymethylcellulase activity were thenvisualized by active staining with Congo red. Namely, the slab gel afterPAGE was placed on the surface of a CMC-agar plate and left over for 30minutes at 37° C. in accordance with the method of Beguin [P. Beguin,Anal. Biochem., 131, 333(1983)]. The CMC-agar plate was composed of 2%(w/v) of CMC (Sunrose A10MC), 3% (w/v) of NaCl, 10% (v/v) of 0.5Mglycine-sodium hydroxide buffer (pH: 9.0) and 0.8% (w/v) of agar(product of Difco). This agar plate was stained with a 0.1% (w/v) Congored (product of Wako Pure Chemical Industries, Ltd.) solution for 15minutes at room temperature and then destained with a 1.0M NaCl solutionfor 15 minutes. As a result, a protein band havingcarboxymethylcellulase activity was detected at a position of 0.45 interms of relative mobility (Rm).

Example 5

With respect to the crude enzyme solution obtained in Example 3, theisoelectric point (pI) was determined by PAGE in accordance with themethod of Wringley [C. W. Wringley, Methods Enzymol., 22, 559(1971)].One hundred microliter of each enzyme solution (1 mg/ml) waselectrophoresed in a 5% (w/v) acrylamide gel column containing 13.3(w/v) of glycerol and 6.3% (v/v) of Pharmalyte (product of Pharmacia; pHrange: 3-10) at a constant voltage of 200 V for about 15 hours. Thecathode and anode were connected to an upper chamber (20 mM H₃ PO₄) anda lower chamber (0.1M sodium hydroxide), respectively. After PAGE, theproteins were visualized by staining with 0.2% (w/v) Coomassie brilliantblue R-250 (product of Merck). Protein bands associated withcarboxymethylcellulase activity were visualized by active staining inthe same manner as in Example 4 to measure the relative mobility of anactive band and to determine a pI value from a standard curve of knownproteins. As a result, it was found to be pH 4.5.

Example 6

About 0.5 g of a soil sample, which had been collected in Los Banos,Laguna, The Philippines, was suspended in 9 ml of a sterilephysiological saline solution and heated for 10 minutes at 80° C. Thethus-heat-treated soil sample was suitably diluted, spread on an agarplate [PCMC medium (Medium A)] and then incubated for 3 days at 30° C.After the incubation, colonies with clear zones were spread on the sameagar plate to incubate them for 3 days at 30° C., thereby formingcolonies again.

Bacillus sp. PKM-5812 (International Deposition No. BP-4088) accordingto this invention was obtained in accordance with the above-describedprocedure.

Example 7

A portion of Bacillus sp. PKM-5812 obtained in Example 6 was subjectedto slant culture using Medium B (prepared in the same manner as inMedium A except for omission of Trypan blue dye). Colonies thus obtainedwere then inoculated into 5 ml of a CMC-peptone medium (Medium C)contained in a 50-ml test tube and incubated for 2 days at 30° C. whileshaking at 300 rpm. A 1-ml portion of the above culture was furtherinoculated into the 50-ml portion of the Medium C contained in a 500-mlSakaguchi flask and incubated for 3 days at 30° C. while shaking at 120rpm.

Cells were removed from the culture medium thus obtained bycentrifugation to obtain a supernatant containing Carboxymethylcellulase5812.

Example 8

The supernatant obtained in Example 7 was treated with ammonium sulfate(pH was kept neutral with dilute aqueous ammonia), and proteins whichprecipitated at 90% saturation were collected by centrifugation(11,000×g). The precipitate was dissolved in 20 mM phosphate buffer ofpH 7.0, and dialyzed overnight at 5° C. against the same buffer.

The concentration of the proteins was determined by means of a Bio-Radprotein assay kit (product of Bio-Rad Laboratories) making use of bovineplasma gamma globulin as a standard in accordance with the method ofBradford [M. Bradford, Anal. Biochem., 72, 248(1976)], whereby a crudeenzyme solution having a specific activity of 7.73 unit (U)/mg ofprotein was found to be obtained.

Example 9

With respect to the crude enzyme solution obtained in Example 8,polyacrylamide gel electrophoresis (PAGE) was carried out on 9% (w/v)polyacrylamide slab gel (70 mm×80 mm, 1.0 mm thickness) making use of150 mM tris-glycine buffer (pH: 8.8) as a running buffer in accordancewith the method of Davis [B. J. Davis, Ann. N. Y. Acad. Sci., 121,404(1964)]. The electrophoresis was performed at 10 mA per gel for about2 hours at room temperature. The proteins were then visualized by silverstaining (kit of silver stain, product of Kanto Chemical Co., Inc.) inaccordance with the method of Oakey et al. [B. R. Oakley, D. R. Kirschand N. R. Morris, Anal. Biochem., 105, 361(1980)].

Protein bands associated with carboxymethylcellulase activity were thenvisualized by active staining with Congo red. Namely, the slab gel afterPAGE was placed on the surface of a CMC-agar plate and left over for 30minutes at 37° C. in accordance with the method of Beguin [P. Beguin,Anal. Biochem., 131, 333(1983)]. The CMC-agar plate was composed of 2%(w/v) of CMC (Sunrose A10MC), 3% (w/v) of NaCl, 10% (v/v) of 0.5Mglycine-sodium hydroxide buffer (pH: 9.0) and 0.8% (w/v) of agar(product of Difco). This agar plate was stained with a 0.1% (w/v) Congored (product of Wako Pure Chemical Industries, Ltd.)solution for 15minutes at room temperature and then destained with a 1.0M NaCl solutionfor 15 minutes. As a result, a protein band havingcarboxymethylcellulase activity was detected at a position of 0.43 interms of relative mobility (Rm).

Example 10

With respect to the crude enzyme solution obtained in Example 8, theisoelectric point (pI) was determined by PAGE in accordance with themethod of Wringley [C. W. Wringley, Methods Enzymol., 22, 559(1971)].One hundred microliter of each enzyme solution (1 mg/ml) waselectrophoresed in a 5% (w/v) acrylamide gel column containing 13.3(w/v) of glycerol and 6.3% (v/v) of Pharmalyte (product of Pharmacia; pHrange: 3-10) at a constant voltage of 200 V for about 15 hours. Thecathode and anode were connected to an upper chamber (20 mM H₃ PO₄) anda lower chamber (0.1M sodium hydroxide), respectively. After PAGE, theproteins were visualized by staining with 0.2% (w/v) Coomassie brilliantblue R-250 (product of Merck). Protein bands associated withcarboxymethylcellulase activity were visualized by active staining inthe same manner as in Example 9 to measure the relative mobility of anactive band and to determine a pI value from a standard curve of knownproteins. As a result, it was found to be pH 4.5.

Industrial Applicability

The enzymes according to this invention are broad in active pH range andactive temperature range and are not inhibited by formulationingredients for detergents, such as various surfactants, metal ions andproteinases. Therefore, they can be advantageously used as formulationcomponents for detergent compositions.

We claim:
 1. Carboxymethylcellulase 5430 having the followingphysicochemical properties:(1) Action:The carboxymethylcellulase 5430can hydrolyze carboxymethylcellulose; (2) Substrate specificity:Thecarboxymethylcellulase 5430 shows strong activity towardcarboxymethylcellulose and lichenan, and also is active toward cellulosepowder, phosphoric acid-swollen cellulose, cellobiose,p-nitrophenol-β-D-glucopyranoside and p-nitrophenyl-β-D-cellobioside;(3) Active pH range and optimum pH:The carboxymethylcellulase 5430 isactive in a pH range of 4.2-12.5 and has two optimum pH values of about7.7 and about 9.5; (4) pH Stability:Even when kept overnight at 5° C.,the carboxymethylcellulase 5430 is scarcely deactivated over a pH rangeof 4.6-12.8; (5) Active temperature range and optimum temperature:Thecarboxymethylcellulase 5430 is active over a temperature range of10°-78° C., and its optimum temperature is at about 55° C.; (6) Thermalstability:The carboxymethylcellulase 5430 is scarcely deactivated evenafter being heated for 10 minutes at 60° C., and still retains someactivity even after being heated to 80° C.; (7) Effects ofsurfactants:The activity of the carboxymethylcellulase 5430 is notinhibited by sodium dodecyl sulfate (SDS), sodium linearalkylbenzenesulfonate (LAS), sodium alkyl sulfate (AS), sodiumpolyoxyethylene alkyl sulfate (ES), sodium α-olefin sulfonate (AOS),sodium α-sulfonated fatty acid ester (α-SFE), fatty acid salt (soap) andpolyoxyethylene alkyl ether; (8) Molecular weight:The molecular weightof the carboxymethylcellulase 5430 is 26,000±1,000 as estimated by gelfiltration on Bio-Gel A 0.5 m, wherein said carboxymethylcellulase 5430is isolated from Bacillus sp. PKM-5430 (FERM BP-4087).